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Resistance to Tgfβ Suppression and Improved Anti-Tumor Responses In ARTICLE DOI: 10.1038/s41467-017-01427-1 OPEN Resistance to TGFβ suppression and improved anti- tumor responses in CD8+ T cells lacking PTPN22 Rebecca J. Brownlie 1,2, Celine Garcia1, Mate Ravasz1, Dietmar Zehn3, Robert J. Salmond 1,2 & Rose Zamoyska 1 Transforming growth factor β (TGFβ) is important in maintaining self-tolerance and inhibits T cell reactivity. We show that CD8+ T cells that lack the tyrosine phosphatase Ptpn22, a major 1234567890 predisposing gene for autoimmune disease, are resistant to the suppressive effects of TGFβ. Resistance to TGFβ suppression, while disadvantageous in autoimmunity, helps Ptpn22−/− T cells to be intrinsically superior at clearing established tumors that secrete TGFβ. Mechanistically, loss of Ptpn22 increases the capacity of T cells to produce IL-2, which overcomes TGFβ-mediated suppression. These data suggest that a viable strategy to improve anti-tumor adoptive cell therapy may be to engineer tumor-restricted T cells with mutations identified as risk factors for autoimmunity. 1 Institute of Immunology and Infection Research, University of Edinburgh, Ashworth Laboratories, Charlotte Auerbach Road, Edinburgh EH9 3FL, UK. 2 Leeds Institute of Cancer and Pathology, University of Leeds, Wellcome Trust Brenner Building, St James’s University Hospital, Leeds LS9 7TF, UK. 3 School of Life Sciences Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, Freising 85354, Germany. Correspondence and requests for materials should be addressed to R.J.S. (email: [email protected]) or to R.Z. (email: [email protected]) NATURE COMMUNICATIONS | 8: 1343 | DOI: 10.1038/s41467-017-01427-1 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01427-1 cell immune tolerance is maintained in the periphery by a response to both strong and weak antigens, the latter being a Tcombination of extrinsic factors, including the action of common trait of tumor-associated antigens, which can otherwise regulatory T cells (Treg) and suppressive cytokines such as limit robust anti-tumor immune responses. These results suggest transforming growth factor β (TGFβ). TGFβ cytokines are posi- that targeting genes associated with susceptibility to auto- tive and negative regulators of proliferation and differentiation in immunity may be a viable strategy to improve the efficacy of many cell lineages, and play a key role in maintaining peripheral adoptive T cell immunotherapy. T cell tolerance1. TGFβ signaling was shown to be essential to 2–5 maintain peripheral T cell quiescence in vivo and to be a Results negative regulator of T cell proliferation in vitro6. Mice whose −/− Ptpn22 T cells resist TGFβ-mediated suppression.TGF-β T cells are unresponsive to TGFβ signals either by expressing a was shown previously to suppress low-affinity T cell responses more dominant negative TGFβRII transgene2,4, or through specificT effectively than high-affinity responses8 and we showed a similar cell deletion of the TGFβ receptor, Tgfbr13,5 die from fatal lym- role for PTPN22 in limiting CD8+ T cell responses18. Using the phoproliferative disease. In the absence of TGFβ signaling, per- − − OVA-specific TCR transgenic mouse, OT-1 on a Rag-1 / back- ipheral CD4+ and CD8+ T cells become activated in a cell- ground (hereafter called OT-1 T cells), for which a number of intrinsic fashion, most likely due to homeostatic expansion from peptides have been characterized, which span a range of affinities19, the lymphopenic environment shortly after birth, and develop − − we examined the sensitivity of control and Ptpn22 / OT-1 T cells effector function and pathogenicity7,8. Thus, TGFβ signals are to inhibition by TGFβ in vitro. particularly important for the regulation of the TCR-mediated, As reported previously8, TGFβ markedly inhibited antigen- weak, self-ligand interactions that maintain peripheral T cell induced proliferation of OT-1 TCR transgenic T cells in a dose- homeostasis. dependent manner (Fig. 1a–c). This suppression occurred TGFβ signaling is important also in restricting T cell responses following stimulation both with strong agonist, SIINFEKL (N4) to tumors9,10. In the context of CD8+ T cells, TGFβ targets the peptide and with weak agonist, SIITFEKL (T4) peptide. Enhanced transcription of key effector molecules and interferes with their − − proliferation of Ptpn22 / OT-1 T cells compared to control OT- ability to kill tumor cells10. These observations have led to the 1 cells was particularly apparent in response to weak agonist suggestion that targeting TGFβ sensitivity, for example, by inac- peptide, T4, in the absence of TGFβ (Fig. 1a–c), as we reported tivating the TGFβ receptor in T cells, could be beneficial in anti- previously18. Significantly, both N4- and T4-induced proliferation tumor T cell therapy11. − − of Ptpn22 / OT-1 T cells were extremely refractory to TGFβ There are several parallels between the induction of T cell inhibition (Fig. 1a–c) compared to control OT-1 T cells. In autoimmunity and effective tumor immunosurveillance12,13. − − response to N4 peptide, no inhibition of Ptpn22 / OT-1 T cell T cells in autoimmune disease display functional characteristics proliferation was seen at doses of up to 5 ng/ml TGFβ, while in that, in a tumor setting, may be beneficial. For example, auto- − − response to T4 peptide, Ptpn22 / OT-1 T cells required ~10-fold reactive T cells respond effectively to poorly immunogenic anti- higher concentrations of TGFβ than control OT-1 T cells to show gens and are resistant to immune-regulatory mechanisms2,8. equivalent suppression of proliferation. Similarly, in order to mount effective anti-cancer responses, The mechanism of TGFβ-mediated suppression of T cell T cells need to overcome a strongly inhibitory tumor micro- responses is not well understood. PTPN22 is known to target environment, characterized by the presence of Tregs and high signaling molecules immediately downstream of the TCR, such as levels of inhibitory cytokines such as TGFβ. Indeed, TGFβ is − − Lck and ZAP70, so it was unclear why Ptpn22 / T cells would be expressed by a wide range of cancer cell types (https://portals. more resistant than control T cells to TGFβ-mediated suppres- broadinstitute.org/ccle/home) as well as by tumor-infiltrating sion. We asked at what point TGFβ suppression acted to inhibit immune cells such as myeloid-derived suppressor cells14. control OT-1 T cells. Time-course analysis demonstrated that Genome-wide association studies have shown that the hema- TGFβ inhibition of OT-1 T cell proliferation was apparent at day topoietic tyrosine phosphatase, PTPN22, has a commonly 3 (Fig. 1d), but in order to achieve this, TGFβ needed to be expressed variant, PTPN22R620W, that is highly associated with present from the start of culture. Delaying addition of TGFβ by predisposition to a number of autoimmune diseases15,16. PTPN22 2 days failed to limit antigen-induced proliferation (Fig. 1e). regulates Ag-specific T- and B-cell responses and some pattern Similarly, TGFβ-mediated inhibition of OT-1 T cell proliferation recognition signaling pathways in myeloid cells that lead to could be alleviated by an inhibitor to the TGFβ receptor, but only production of type 1 interferons15. We showed previously that − − when added from the beginning of culture (Fig. 1f). mouse Ptpn22 / T cells are partially resistant to wild-type Treg- These data show that TGFβ acted early in T cell activation, as mediated suppression17 and are more responsive to low-affinity by 48 h after stimulation cells were resistant to the anti- antigens18. Given the critical dual role of TGFβ in limiting proliferative effects of TGFβ. Accordingly, we analyzed activation autoimmune and anti-tumor responses, we investigated the marker and transcription factor expression at 24 h following impact of PTPN22 deficiency on the responses of CD8+ T cells to stimulation of OT-1 T cells. In control OT-1 T cells, TGFβ this key inhibitory cytokine. Furthermore, we sought to test the limited the extent of weak agonist T4-induced upregulation of the hypothesis that PTPN22 acts as a brake to limit the effectiveness − − IL-2 receptor-α chain (CD25), and key transcription factors Myc of anti-tumor T cell responses. We show that Ptpn22 / CD8 − − and IRF4, but less so Tbet, whereas Ptpn22 / OT-1 T cells were T cells are considerably more resistant to the suppressive effects refractory to these inhibitory effects of TGFβ (Fig. 2a, b). These of TGFβ than WT T cells. Concentrations of TGFβ that suppress data indicate that loss of PTPN22 counteracted TGFβ inhibition proliferation and differentiation of effector cytokines in WT − − during the early phase of TCR engagement. T cells show little inhibition of these processes in Ptpn22 / CD8 T cells. Upon stimulation with both weak and strong agonists − − −/− peptides Ptpn22 / CD8 T cells produce more IL-2 than WT Ptpn22 CD8+ T cells are superior at tumor clearance. The − − T cells and IL-2 interferes with the suppressive effect of TGFβ. resistance of Ptpn22 / T cells to TGFβ-mediated suppression − − Consequently upon adoptive transfer, Ptpn22 / CD8 T cells are in vitro led us to ask whether these CD8+ T cells would be more better able than WT CD8 T cells to control the growth of effective for adoptive cell therapy against TGFβ-secreting tumors − − established tumors that secrete TGFβ. Importantly, the superior in vivo. First, we evaluated the ability of control and Ptpn22 / − − anti-tumor capacity of Ptpn22 / CD8+ T cells is observed in OT-1 T cells to reject EL4 lymphoma cells (EL4-OVA) expressing 2 NATURE COMMUNICATIONS | 8: 1343 | DOI: 10.1038/s41467-017-01427-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01427-1 ARTICLE abc0 β N4 N4 TGF (ng/ml) Control 2.00 Control 5 4 –/– –/– Ptpn22 1.00 Ptpn22 N4 T4 3 100 100 0.50 80 80 2 0.25 0.12 Control 60 60 1 0.06 40 40 0 0.03 5 5 0 20 20 0 0.2 0.6 1.7 0.2 0.6 1.7 0.06 0 0 0.06 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 T4 T4 100 100 4 1.00 /ml) 0.50 80 80 3 6 –/– 0.25 Ptpn22 60 60 2 0.12 40 40 0.06 20 20 1 Division index 0.03 Cell no.
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